Generation of iPSCs from genetically corrected Brca2 hypomorphic cells: implications in cell reprogramming and stem cell therapy.

Fanconi anemia (FA) is a complex genetic disease associated with a defective DNA repair pathway known as the FA pathway. In contrast to many other FA proteins, BRCA2 participates downstream in this pathway and has a critical role in homology-directed recombination (HDR). In our current studies, we have observed an extremely low reprogramming efficiency in cells with a hypomorphic mutation in Brca2 (Brca2(Δ) (27/) (Δ27)), that was associated with increased apoptosis and defective generation of nuclear RAD51 foci during the reprogramming process. Gene complementation facilitated the generation of Brca2(Δ) (27/) (Δ27) induced pluripotent stem cells (iPSCs) with a disease-free FA phenotype. Karyotype analyses and comparative genome hybridization arrays of complemented Brca2(Δ) (27/) (Δ27) iPSCs showed, however, the presence of different genetic alterations in these cells, most of which were not evident in their parental Brca2(Δ) (27/) (Δ27) mouse embryonic fibroblasts. Gene-corrected Brca2(Δ) (27/) (Δ27) iPSCs could be differentiated in vitro toward the hematopoietic lineage, although with a more limited efficacy than WT iPSCs or mouse embryonic stem cells, and did not engraft in irradiated Brca2(Δ) (27/) (Δ27) recipients. Our results are consistent with previous studies proposing that HDR is critical for cell reprogramming and demonstrate that reprogramming defects characteristic of Brca2 mutant cells can be efficiently overcome by gene complementation. Finally, based on analysis of the phenotype, genetic stability, and hematopoietic differentiation potential of gene-corrected Brca2(Δ) (27/) (Δ) (27) iPSCs, achievements and limitations in the application of current reprogramming approaches in hematopoietic stem cell therapy are also discussed.

Immunoresponse against the transgene limits hematopoietic engraftment of mice transplanted in utero with virally transduced fetal liver.

In utero cell and gene therapies constitute alternative strategies to the postnatal treatment of inherited diseases. Fetal hematopoietic progenitors could be a potential source of donor cells for these strategies. In this study, hematopoietic lineage-negative fetal liver cells from 14.5-day-old fetuses were transduced under different cytokine and culture combinations using a lentiviral vector expressing the enhanced green fluorescent protein (EGFP). When cells were transduced for 6 h in the presence of mSCF, hTPO and FLT3-L in retronectin-coated dishes at a multiplicity of infection of 10 transduction units/cell, up to 70% of granulo-macrophage colony-forming cells expressed the EGFP reporter gene. In utero transplantation experiments revealed that conditions leading to high transduction efficiencies were associated with poor engraftments of syngeneic recipients. Significantly, this effect was associated with the detection of a humoral and cellular immunoresponse against the transgenic protein. Moreover, the humoral response against EGFP was detected not only in in utero transplanted recipients but also in the operated mothers, suggesting the maternal origin of the anti-EGFP immunoresponse. These observations reinforce the necessity of carefully studying the potential immunoresponses in future prenatal gene therapy protocols.

Molecular Evidence of Genome Editing in a Mouse Model of Immunodeficiency.

Genome editing is the introduction of directed modifications in the genome, a process boosted to therapeutic levels by designer nucleases. Building on the experience of ex vivo gene therapy for severe combined immunodeficiencies, it is likely that genome editing of haematopoietic stem/progenitor cells (HSPC) for correction of inherited blood diseases will be an early clinical application. We show molecular evidence of gene correction in a mouse model of primary immunodeficiency. In vitro experiments in DNA-dependent protein kinase catalytic subunit severe combined immunodeficiency (Prkdc scid) fibroblasts using designed zinc finger nucleases (ZFN) and a repair template demonstrated molecular and functional correction of the defect. Following transplantation of ex vivo gene-edited Prkdc scid HSPC, some of the recipient animals carried the expected genomic signature of ZFN-driven gene correction. In some primary and secondary transplant recipients we detected double-positive CD4/CD8 T-cells in thymus and single-positive T-cells in blood, but no other evidence of immune reconstitution. However, the leakiness of this model is a confounding factor for the interpretation of the possible T-cell reconstitution. Our results provide support for the feasibility of rescuing inherited blood disease by ex vivo genome editing followed by transplantation, and highlight some of the challenges.

In vitro and in vivo expression of human erythrocyte pyruvate kinase in erythroid cells: a gene therapy approach.

Human pyruvate kinase deficiency (PKD), an autosomal recessive disorder produced by mutations in the PKLR gene, is the most common cause of chronic nonspherocytic hemolytic anemia. Transduction of wild-type erythroid (R-type) pyruvate kinase (RPK) cDNA into deficient hematopoietic stem cells could be of potential use as rescue therapy in severe clinical cases. In this study, gammaretroviral vectors expressing human RPK were designed as possible gene therapy candidates for this disease. Through real-time quantitative reverse transcriptase-polymerase chain reaction, Western blotting, and flow cytometric analysis, we demonstrate stable RPK expression in both undifferentiated and differentiated murine erythroleukemia cells. In this in vitro assay, the proportion of transduced cells and the intensity of expression of the transgene remained unaltered after 6 months of culture. Moreover, transplanting human RPK-transduced Lin(-)Sca-1(+) mouse cells in myeloablated primary and secondary recipients rendered high proportions of erythroid precursors and mature erythrocytes expressing RPK, without inducing hematopoietic effects. These findings suggest that retroviral vectors could be useful for the delivery and expression of RPK in erythroid cells, and provide evidence of the potential use of gene therapy strategies to phenotypically correct erythroid PKD.

A simplified approach to improve the efficiency and safety of ex vivo hematopoietic gene therapy in fanconi anemia patients.

Fanconi anemia (FA) is an inherited DNA repair disorder characterized by genetic instability of cells lacking a functional FA/BRCA pathway. Previous studies have shown that in vitro stimulation of bone marrow cells (BMCs) from FA mice promotes apoptosis, reduces the reconstitution ability of the stem cells, and induces myelodysplasia and myeloid leukemia upon reinfusion of the cells. This suggests the convenience of adapting standard protocols of gene therapy to FA. Here we show that the reserve of BM progenitors in FA patients is generally below 20% of normal values. Because this reduced reserve could activate the cycling of BM progenitors, we developed a simplified protocol to transduce BMCs from FA patients with gammaretroviral vectors. We demonstrate that a short in vitro manipulation (12-24 hr) of fresh mononuclear BMCs is sufficient to transduce 42% of hematopoietic progenitors from FA-A patients, in the absence of in vitro prestimulation. When FANCA-expressing vectors were used, this simple procedure reversed the phenotype of the BM progenitors from these patients. We propose that our approach will be more efficient and safer compared with standard gene therapy protocols for FA.

Engraftment kinetics of human CD34+ cells from cord blood and mobilized peripheral blood co-transplanted into NOD/SCID mice.

We have reported short periods of post transplant neutropenia in human patients co-transplanted with cord blood (CB) and low numbers of haploidentical mobilized peripheral blood (MPB) CD34+ cells. To investigate the effect that the proportion of MPB to CB cells may have on engraftment kinetics, we have co-transplanted fixed numbers of human CB CD34+ cells mixed with different numbers of MPB CD34+ cells into NOD/SCID mice. We periodically quantified the proportion of human cells and the relative contribution of MPB and CB cells to the human engraftment on marrow aspirates. At the lowest MPB/CB ratios (5 : 1, 10 : 1), the contribution of CB cells predominated at all time points analyzed, and in three out of four experiments MPB cell contributions progressively decreased from day +15. At higher MPB/CB ratios, MPB cells had a more important contribution to both early and late engraftment, with the highest cell ratio resulting in only marginal CB cell engraftment. Therefore, our results showed greater potential, on a per cell basis, of human CB vs MPB cells for competitive sustained engraftment in the xenogeneic model used, which was only abrogated by the co-infusion of very high numbers of MPB cells.

Transplantation of syngenic bone marrow contaminated with NGFr-marked WEHI-3B cells: an improved model of leukemia relapse in mice.

With the aim of developing a model mimicking the relapse of patients transplanted with leukemia-contaminated grafts, myelomonocytic leukemia WEHI-3B D+ cells were first transduced with a retroviral vector encoding the low-affinity human nerve growth factor receptor (NGFr). Clones with a stable and homogeneous expression of the transgene and with a similar in vitro behavior to the parental cell line were selected for further experiments. The analysis of bone marrow (BM) contaminated with WEHI-3B/NGFr cells revealed a linear correlation (r2 = 0.999) between the actual values of BM contamination and the experimental data determined by flow cytometry. Balb/c mice were myeloablated and transplanted with syngenic BM contaminated with graded numbers of leukemic cells; dose-dependent survival curves were obtained, regardless of whether parental or WEHI-3B/NGFr cells were infused. The leukemia dissemination in recipients transplanted with WEHI-3B/NGFr contaminated grafts was easily determined by means of simple flow cytometry analysis of the NGFr marker. A leukemia dose-dependent increase in the number of PB leukocytes was observed in transplanted recipients at 20 days post-transplantation with no changes in myelomonocytic cells. As deduced from our observations, the transplantation of syngenic BM contaminated with WEHI-3B/NGFr cells constitutes an improved model of autograft-mediated leukemia relapse and a good tool for studies of leukemia cell purging.

Cytotoxic chemotherapy regimens that increase dose per cycle (dose intensity) by extending daily dosing from 5 consecutive days to 28 consecutive days and beyond.

Dose intensity, defined as dose administered per unit time, has emerged as a potentially important measurement of anticancer drug exposure and determinant of efficacy. There are several strategies for increasing dose intensity, one being a protracted daily dosing strategy without major dose reduction for toxicity. This strategy involves continued therapy during periods of recovery from reversible toxicity, and it inherently challenges our understanding that renewing tissues cannot repopulate (recover) in the continued presence of cytotoxic drug. We have tested this idea directly in a murine preclinical trial. Specifically, we have tested whether acutely myelotoxic doses of gemcitabine (i.p. injection, 6.0 mg/m2/day), acetyldinaline [CI-994; GOE 5549; PD 123 654; 4-acetylamino-N-(2'-aminophenyl)-benzamide, 150 mg/m2/day p.o.], and/or melphalan (i.p. injection, 7.2 mg/m2/day) can be tolerated for 28 consecutive days and whether suppressed bone marrow function recovers despite this protracted daily therapy. The three drugs all caused acute neutropenia and suppression of medullary hematopoiesis. Damage to progenitor populations exposed to acetyldinaline and gemcitabine was not as severe as that caused by melphalan, in which case absolute neutrophil count, mature progenitors (colony-forming unit granulocyte/macrophage), and immature progenitors (colony-forming unit-S) progressively declined to severely depressed levels. Marrow recovery was observed during continued daily treatment with acetyldinaline and gemcitabine but not melphalan, and marrow function completely recovered after finishing the 28-day course. Pharmacology studies proved that protracted therapy causes little, if any, change in cellular drug tolerance or systemic exposure.

Involvement of the bone marrow stroma in the residual hematopoietic damage induced by irradiation of adult and young mice.

The analysis of femoral hematopoiesis of mice irradiated with 7 Gy x-rays at the age of 1 and 12 weeks evidenced a differential hematopoietic dysfunction in the long term. Significant hematopoietic damage was observed 1 year after irradiation of 12-week-old mice: a marked reduction in the number of hematopoietic progenitors and a severe impairment in the self-renewal capacity of the colony-forming unit-spleen (CFU-S) population. In the case of the 1-week-old irradiated mice, normal values in the femoral hematopoietic progenitors were observed, although a significant impairment in the capacity for self-renewal of the CFU-S population was apparent. The role that the stromal cells played in these hematopoietic failures was investigated by ectopic implantation of bone marrow from the irradiated animals into the renal capsule of nonirradiated mice. Bone marrow implants from both types of donor-irradiated mice developed ossicles that were incapable of sustaining normal values of host hematopoietic progenitors, thus indicating that radiation mediated long-term damage in the hematopoietic lodging capacity of the stromal cells. However, analysis of the number of CFU-S generated per ossicle-derived spleen colony revealed that the irradiation of hematopoietic stromas resulted in an improved self-renewal capacity of lodged unirradiated CFU-S precursors. Our data strongly suggest that the impairment in the long-term self-renewal capacity of the CFU-S population is not a result of the stromal damage. Rather, this stromal damage might actually generate a stimulatory response facilitating the preservation of the low numbers of primitive precursors that survived the irradiation.

Residual damage of lymphohematopoietic repopulating cells after irradiation of mice at different stages of development.

OBJECTIVE: The purpose of this study was to evaluate the repopulating properties of bone marrow (BM) from mice irradiated during embryonic and adult stages of development. MATERIALS AND METHODS: Four-day-old embryos, 17-day-old fetuses, and 12-week-old mice were irradiated with of 1 or 3 Gy of x-rays. At 3 and 9 months postirradiation, the effects generated within the different compartments of repopulating cells (RCs) were evaluated by determining, in a BM competition assay, the contribution of the irradiated precursors to the lymphohematopoiesis of recipients at different times posttransplantation (3, 9, and 15 months). RESULTS: The irradiation of 4-day-old embryos with either 1 or 3 Gy did not produce residual repopulation or differentiation effects within the different RCs assayed. However, significant impairments in RC functionality were observed in mice irradiated on the 17th day postconception or at the 12th week of age. Whereas irradiation of these animals with 1 Gy did not impair the long-term functionality of the very primitive 15-month-old RCs, irradiation with 3 Gy generated sustained impairment in all tested types of hematopoietic progenitors and RCs. Moreover, repopulation data derived from the analysis of recipient BM and thymus strongly suggested that the observed effects were produced within the multipotent pool of lymphohematopoietic RCs. CONCLUSIONS: Our data show the generation of long-term effects in the multipotent RCs of mice irradiated at fetal and adult stages of growth and reveals the normal functionality of the RCs from animals irradiated during the early stages of embryonic development.

Restrictions in the stem cell function of murine bone marrow grafts after ex vivo expansion of short-term repopulating progenitors.

We investigated the in vivo implications associated with the ex vivo expansion of 5-fluorouracil (5-FU) preactivated murine bone marrow (BM) grafts. Analysis of cultures established with BM cells collected 2 and 4 days after 5-FU treatment (2d and 4d 5-FU BM, respectively) and stimulated with IL-3 + IL-6 and IL-3 + SCF resulted in the generation of samples highly enriched for colony-forming units granulocyte/macrophage (CFU-GMs). This result was best shown in cultures established with 4d 5-FU BM and incubated for 3 days with IL-3 + SCF; in these samples up to 10% of the cellularity consisted of CFU-GMs. Analyses of the spleen colony-forming unit (CFU-S)12/CFU-S8 ratio revealed a continuous decline in this parameter during the expansion process, suggesting a predominant differentiation stimulus in the cultures. Transplantation of BM grafts into myeloablated recipients revealed a marked improvement in the short-term radioprotection capacity (30 days survival) of ex vivo expanded BM, which was most significant in the case of 3-day expanded grafts. In contrast to this finding, progressive impairment of the long-term radioprotection capacity of the grafts was found to be associated with the expansion process. Irrespective of the type of ex vivo manipulation used, a predominantly lymphohematopoietic repopulation by donor cells was observed in all recipients analyzed in the long term (4-7 months) posttransplantation. To investigate more thoroughly whether the repopulation ability of the grafts was modified to some extent during the ex vivo expansion process, BM competition assays were performed. The data obtained at 30 and 120 days posttransplantation indicated that under the best conditions assayed (4d 5-FU BM expanded for 3 days with IL-3 + SCF) almost no change in the competitive repopulation ability of the grafts was produced. However, when analysis was delayed to 300 days posttransplantation, a twofold reduction in the stem cell function of the expanded grafts was noted. Based on this data it is proposed that, under our experimental conditions, a significant expansion in the number of short-term repopulating progenitors is produced concomitantly with a differentiation stimulus of the culture, which moderately restricts the number and/or the longevity of the self-renewing stem cells.

In vitro and in vivo production of CFU-S-stimulating activities after 5-Gy total body irradiation.

In vitro and in vivo production of CFU-S-stimulating activities have been described after 5-Gy irradiation of mice using a diffusion chamber-incubation technique. The experimental results showed that bone marrow obtained 15 min after 5 Gy total body irradiation did not present detectable CFU-S stimulating activity, but was able to produce such activity in vitro within 20 h. Once the activity had been produced, 2.5 h was enough to stimulate the proliferation of CFU-S. When CFU-S were incubated for 20 h in the peritoneal cavities of mice that had been irradiated with 5 Gy immediately prior to the insertion of the chambers, or five days earlier, a marked stimulation of CFU-S turnover was observed, demonstrating the in vivo production of some humoral activity capable of stimulating CFU-S.

In vitro and in vivo susceptibility of mouse megakaryocytic progenitors to strain i of parvovirus minute virus of mice.

OBJECTIVE: Intranasal inoculation of the i strain of the parvovirus minute virus of mice (MVMi) into immunodeficient SCID mice induces suppression of myeloid and erythroid progenitors in the bone marrow (BM) and lethal leukopenia. In the present study, we investigated whether the mouse megakaryocytic lineage was susceptible to MVMi. MATERIALS AND METHODS: In vitro and in vivo infections with purified MVMi were conducted and their effects on the megakaryocytic lineage studied. RESULTS: In vitro infection of BM cells showed a multiplicity of infection-dependent inhibition in the colony-forming ability of megakaryocytic progenitors (colony-forming unit megakaryocyte [CFU-MK]). Neutralization or heat inactivation of the virus abrogated this inhibition. Expression of the MVMi nonstructural-1 protein was detected in the in vitro infected and cultured megakaryocytic cells. In vivo, intranasal inoculation of a lethal dose of virus was incapable of producing significant thrombocytopenia, although an increase in mean platelet volume was observed. Significantly, in the BM of these animals, a progressive decrease in CFU-MK was noted from day 14 postinfection, with survival rates less than 1% by day 35 postinfection. At day 35 postinfection, intermediate megakaryocytic differentiation stages showed maintenance of the proportion and ploidy of cells and a moderate decrease in the total number of these cells per femoral BM. CONCLUSIONS: The results demonstrate that MVMi is capable of inhibiting the proliferative capacity of megakaryocytic committed progenitors both in vitro and in vivo. Moreover, the in vivo data show that depletion of BM CFU-MK is compensated by the system, and platelet counts in the peripheral blood are maintained close to normal values.

Latent hematopoietic stem cell toxicity associated with protracted drug administration.

OBJECTIVE: The protracted administration of near-conventional daily doses of chemotherapeutic agents is a strategy to increase dose intensity and, potentially, efficacy as well. However, protracted therapy carries the risk of damage to stem cells in proliferative tissues that are not targeted by intermittent schedules. Therefore, we have investigated the effects produced by the protracted administration of two anticancer drugs on hematopoietic stem cell function. MATERIALS AND METHODS: We used the competitive repopulating assay to assess stem cell damage caused by protracted daily drug treatment of mice. RESULTS: Treatment with acetyldinaline for 10 consecutive days mediated a modest effect on the short-term repopulating cells (STRCs) but spared the long-term repopulating cells (LTRCs). Gemcitabine for 10 days led to a modest decline in both the STRCs and LTRCs. Extending treatment with gemcitabine for 28 days resulted in more severe repopulating cell (RC) damage, which was much worse than in acetyldinaline-treated mice. As expected, melphalan for 10 or 28 days mediated a marked reduction in all of the RCs of treated mice. The analysis of the RCs from mice that were allowed a 1-year recovery period after completing the 28-day treatment with either acetyldinaline or gemcitabine showed normal levels of neutrophils and bone marrow (BM) progenitors. However, a reduction in the RCs was observed in both groups, with larger reductions in gemcitabine-treated mice. CONCLUSIONS: Our data show that protracted treatment with gemcitabine, but not acetyldinaline, of mice caused severe permanent damage to the stem cell components. Therefore, although 28-day therapy with acetyldinaline or gemcitabine appeared to be well tolerated at the level of peripheral blood and bone marrow progenitors, gemcitabine produces permanent stem cell damage when used in long-term administration regimens that should perhaps only be explored clinically with stem cell support available.

In vitro toxicity of ET-743 and aplidine, two marine-derived antineoplastics, on human bone marrow haematopoietic progenitors. comparison with the clinical results.

Ecteinascidine-743 (ET-743) and aplidine are two marine-derived antineoplastics currently in phase II development. With the aim of evaluating whether in vitro haematopoietic studies can predict the toxicity of these two drugs in patients, human bone marrow (BM) samples were incubated with these drugs under conditions which mimicked the administration exposures used in the clinics. As it was observed in different cancer cell lines, ET-743 was more toxic on an equimolar basis in human hematopoietic progenitors (inhibitory concentration reducing the viability to 50% after 24 h exposures; IC50(24h): 10-50 nM) compared with doxorubicin (IC50(24h) values: 280-460 nM), used as a control anticancer drug. In contrast to the high haematotoxic effects observed for ET-743, similar IC values were obtained for aplidine (IC50(24h): 150-530 nM) compared with doxorubicin. For both ET-743 and aplidine, the megakaryocytic progenitor was the most sensitive, compared with the other haematopoietic progenitors (IC50 values were 3- to 5-fold lower in the CFU-Megs compared with the CFU-GMs). The observation that the Cmax observed in patients treated with the aplidine maximum tolerated dose (MTD) (7.1 nM) was 21-75 fold lower than the IC50(24h) value observed for the different haematopoietic progenitors is highly consistent with the lack of haematotoxicity observed in patients treated with this drug. In the case of ET-743, differences between the Cmax value corresponding to the MTD (2.6 nM) and the in vitro IC50 values corresponding to the different progenitors were much lower (4-19-fold), also consistent with the haematotoxicity that was observed in patients treated at recommended doses (RDs) and MTDs. Although CFU-Megs were more sensitive than CFU-GM progenitors to ET-743 in vitro, clinical data showed that neutropenic events were more frequent than thrombocytopenic episodes. Aiming to further improve the predictive value of in vitro IC values corresponding to the different haematopoietic progenitors, additional refinement parameters derived from pharmacokinetic and animal studies are proposed.

Pyruvate kinase during in vitro differentiation of GM-CFC haemopoietic precursor in mice: modulation by L-alanine and L-phenylalanine.

Pyruvate kinase modulation by L-alanine and L-phenylalanine was studied in cells obtained from in vitro culture of GM-CFC at different stages of development. L-Alanine and L-phenylalanine exhibit different inhibition behaviour at both differentiation stages and at various PEP concentration ranges. Therefore, a change in PK expression during granulocyte-macrophage development is suggested.

Quantitative PCR analysis reveals a high incidence of large intragenic deletions in the FANCA gene in Spanish Fanconi anemia patients.

Fanconi anaemia is an autosomal recessive disease characterized by chromosome fragility, multiple congenital abnormalities, progressive bone marrow failure and a high predisposition to develop malignancies. Most of the Fanconi anaemia patients belong to complementation group FA-A due to mutations in the FANCA gene. This gene contains 43 exons along a 4.3-kb coding sequence with a very heterogeneous mutational spectrum that makes the mutation screening of FANCA a difficult task. In addition, as the FANCA gene is rich in Alu sequences, it was reported that Alu-mediated recombination led to large intragenic deletions that cannot be detected in heterozygous state by conventional PCR, SSCP analysis, or DNA sequencing. To overcome this problem, a method based on quantitative fluorescent multiplex PCR was proposed to detect intragenic deletions in FANCA involving the most frequently deleted exons (exons 5, 11, 17, 21 and 31). Here we apply the proposed method to detect intragenic deletions in 25 Spanish FA-A patients previously assigned to complementation group FA-A by FANCA cDNA retroviral transduction. A total of eight heterozygous deletions involving from one to more than 26 exons were detected. Thus, one third of the patients carried a large intragenic deletion that would have not been detected by conventional methods. These results are in agreement with previously published data and indicate that large intragenic deletions are one of the most frequent mutations leading to Fanconi anaemia. Consequently, this technology should be applied in future studies on FANCA to improve the mutation detection rate.

Preserved long-term repopulation and differentiation properties of hematopoietic grafts subjected to ex vivo expansion with stem cell factor and interleukin 11.

BACKGROUND: The ex vivo expansion of hematopoietic grafts has been proposed as an efficient procedure for improving the hematological recovery of recipients. The fate of the long-term repopulating cells during the ex vivo manipulation of the graft is, however, a critical issue in ex vivo expansion protocols and ultimately will define the applicability of this new technology in hematopoietic transplants. METHODS: The repopulating ability of mouse hematopoietic samples was determined by means of bone marrow (BM*) competition assays, using congenic strains that express the pan-leukocyte Ly-5.1 and Ly-5.2 antigens. The generation of potential changes in the repopulating properties of human hematopoietic samples subjected to ex vivo expansion was determined by comparing the engraftment of fresh and ex vivo-manipulated CD34+ cord blood cells in irradiated nonobese diabetic/severe-combined immunodeficient (NOD/SCID) mice. RESULTS: Under our optimized conditions of mouse BM incubation (stem cell factor plus interleukin-11, either with or without macrophage inflammatory protein-1alpha or Flt3 ligand), both the short-term and the mid-term repopulating ability of the ex vivo-expanded samples were significantly improved when compared with fresh samples. In the long-term, no changes in the repopulation and differentiation properties of the graft were observed as a result of the ex vivo expansion process. As deduced from the analysis of NOD/SCID mice transplanted with fresh and ex vivo expanded human CD34+ cord blood cells, the in vitro stimulation mediated by SCF/IL-11/FLT3L was capable of preserving the ability of the grafts to repopulate the lympho-hematopoiesis of recipients for at least 3 months. CONCLUSION: These results indicate that under our optimized conditions of ex vivo expansion, the amplification of the hematopoietic progenitors responsible for the short- and mid-term repopulating properties of the graft can take place without compromising the long-term lympho-hematopoietic repopulating properties.

The influence of plasma concentrations of tri-iodothyronine on the acute increases in insulin and muscle protein synthesis in the refed fasted rat.

To examine whether the low plasma levels of triiodothyronine (T3) in fasted rats might limit the recovery of muscle protein synthesis on refeeding, rats were fasted for either 3 or 4 days and refed with or without pretreatment with thyroid hormones. Fasting suppressed T3 levels, plasma insulin and the rate of the translational phase of muscle protein synthesis (KRNA; the rate per unit RNA), especially after the 4-day fast. On refeeding, plasma T3 levels remained low for more than 3 h after the 3-day fast and for more than 8 h after the 4-day fast. Insulin concentrations increased within the first hour of refeeding, eventually achieving supranormal concentrations after the 3-day fast. The KRNA increased within the first hour of refeeding, achieving well-fed control values by 3 h after the 3-day fast or 24 h after the 4-day fast. The increases in KRNA were significantly correlated with the increases in insulin at low insulin concentrations, achieving a plateau value at 150 pmol/l, so that further increases in insulin were not associated with any further increases in protein synthesis. Pretreatment with thyroid hormone induced increased T3 levels which were maintained for up to 8 h of refeeding. This had no effect on the responses of either insulin or protein synthesis to refeeding after the 3-day fast, but did result in an acceleration of the recovery in the KRNA and plasma insulin levels in the rats fasted for 4 days. Analysis of the insulin-KRNA relationship showed no evidence for any increase in the insulin sensitivity of muscle protein synthesis with thyroid pretreatment, the initial stimulation of protein synthesis on refeeding the rats fasted for 4 days reflecting increased insulin secretion. Since in the untreated animals, insulin secretion on refeeding was also correlated with T3 levels, these results are consistent with the previously reported thyroidal dependence of insulin secretion.